We have studied the mechanisms of regulation of m2 and m3 muscarinic and serotonergic 5-HT2 receptors in cultured cerebellar granule cells. Intact granule cells display a much greater selectivity for the muscarinic antagonists AF-DX 116 and 4-DAMP than in crude membranes, while the selectivity pfHHSiD was similar in membranes and whole cells, suggesting that factors which affect antagonist selectivity are lost when cells are disrupted. The muscarinic agonist carbachol down- regulates m2 and m3 receptor mRNAs and proteins in conjunction with an increase in c-Fos mRNA levels. The role of c-Fos protein in the agonist-induced regulation of m2- and m3-muscarinic receptor mRNA is being examined by using cells pretreated with an antisense oligonucleotide to c-Fos mRNA. The agonist-induced down-regulation and antagonist-induced up-regulation of m2- and m3-muscarinic receptor mRNA were accompanied by corresponding changes in the muscarinic receptor proteins, determined by immunoprecipitation with m2- and m3-receptor specific antisera. The 5-HT2 selective serotonergic agonist DOI causes a paradoxical up-regulation of 5-HT2 receptor binding in cerebellar granule cells. Interrupting the signal transduction pathway with pertussis toxin or a dibutyryl phorbol ester failed to block the DOI-induced up-regulation of 5-HT2 receptor binding. The up-regulation could, however, be blocked by cycloheximide, a protein synthesis inhibitor, actinomycin D, an inhibitor of RNA transcription, or colchicine, an inhibitor of mi- crotubule polymerization. The blockade by colchicine was reversible by taxol, a microtubule stabilizer. These observations suggest that the up-regulation of granule cell 5-HT2 receptors is caused by a mechanism independent of the classical GTP binding protein-coupled second messenger system known to be activated by the 5-HT2 receptor. The up-regulation requires synthesis of a protein factor and may in- volve cytoskeletal elements.